Di-(uridine 5′)-tetraphosphate and salts thereof

ABSTRACT

The present invention provides methods for the synthesis of a pharmaceutically useful dinucleotide, P 1 , P 4 -di(uridine 5′)-tetraphosphate, and demonstrates the applicability to the production of large quantities. The methods of the present invention substantially reduce the time required to synthesize diuridine tetraphosphate, for example, to three days or less. The tetrasodium, ammonium, lithium and potassium salts of P 1 , P 4 -di(uridine 5′)-tetraphosphate prepared by these methods are stable, soluble, nontoxic, of high purity and easy to handle during manufacture.

This application is a continuation-in-part of U.S. Application No.09/122,516, filed Jul. 24, 1998, U.S. Pat. No. 6,319,908, which claimsthe benefit of U.S. Provisional Application No. 60/054,147 filed Jul.25, 1997.

TECHNICAL FIELD

This invention relates to methods for the production of therapeuticdinucleotides including novel salts thereof. More specifically, itrelates to methods for synthesis of P¹, P⁴-di(uridine5′)-tetraphosphate, i.e., diuridine tetraphosphate (U₂P₄) which haveadvantages over prior art methods of manufacture.

BACKGROUND OF THE INVENTION

P¹, P⁴-Di(uridine 5′)-tetraphosphate is a dinucleotide of the followingstructure:

wherein:

X is Li, Na, K, NH₄ or H, provided that all X groups are not H.

The free acid of P¹, P⁴-di(uridine 5′)-tetraphosphate, where X ishydrogen, has been previously described as uridine 5′-(pentahydrogentetraphosphate), P′″→5′-ester with uridine (CAS Registry Number:59985-21-6; C. Vallejo et al., Biochimica et Biophysica Acta 438, 305(1976) and H. Coste et al., J. Biol. Chem. 262, 12096 (1987)).

Different methods have been described for the synthesis of purinedinucleotides such as diadenosine tetraphosphate (A₂P₄) (E. Rappaport etal, Proc. Natl. Acad. Sci, 78, 838, (1981); A. Guranowski et al,Biochemistry, 27, 2959, (1988); C. Lobaton et al, Eur. J. Biochem., 50,495, 1975; K. Ng and L. Orgel, Nucl. Acid Res., 15, 3573, (1987)).However, this has not been true for U₂P₄ which is a pyrimidinenucleotide. Although purine nucleotides and pyrimidine nucleotidesappear to be analogous, the methods used for purine nucleotide synthesisdo not necessarily work for pyrimidines such as uridine.

Diuridine tetraphosphate has been shown to have beneficial properties inthe treatment of various diseases, such as chronic obstructive pulmonarydisease (COPD). For example, they have been demonstrated to facilitatethe clearance of mucous secretions from the lungs of a subject such as amammal including humans in need of treatment for various reasons,including cystic fibrosis, chronic bronchitis, asthma, bronchiectasis,post-operative mucous retention, pneumonia, primary ciliary dyskinesia(M. J. Stutts, III, et al, U.S. Pat. No. 5,635,160; PCT InternationalPublication WO 96/40059) and the prevention and treatment of pneumoniain immobilized patients (K. M. Jacobus and H. J. Leighton, U.S. Pat. No.5,763,447). Further therapeutic uses include treatment of sinusitis (PCTInternational Publication WO 98/03177), otitis media (PCT InternationalPublication WO 97/29756), dry eye, retinal detachment, nasolacrimal ductobstruction, the treatment of female infertility and irritation due tovaginal dryness via increased mucus secretions and hydration of theepithelial surface, and enhancing the performance of athletes.

U₂P₄ also has utility as a veterinary product in mammals such as, butnot limited to, dogs, cats and horses.

Prior art methodology describes only one protocol for the production ofdiuridine tetraphosphate. This method is very time consuming, lastingover five days and producing only small amounts of diuridinetetraphosphate (C. Vallejo et al., Biochimica et Biophysica Acta 438,305 (1976), Sillero et al., Eur J Biochem 76, 332 (1972)). According tothis technique, diuridine tetraphosphate was synthesized through areaction of uridine 5′-monophosphomorpholidate (0.54 mmol) with thetriethylamine salt of pyrophosphoric acid (0.35 mmol) in a medium ofanhydrous pyridine (10 ml). After 5 days at 30° C., pyridine was removedfrom the reaction mixture by evaporation, and the residue resuspended inglass-distilled water (8 mL), the suspension applied to a DEAE-cellulosecolumn (37.5×2.6 cm) and fractionated with 3.2 L of a linear gradient(0.06-0.25 M) of ammonium bicarbonate, pH 8.6. The peak eluting between0.17-0.19 M ammonium bicarbonate was partially characterized as U₂P₄ bythe following criteria: insensitivity to alkaline phosphatase,phosphorus to base ratio and analysis of the products of hydrolysis(UTP+UMP), after treatment with phosphodiesterase I, by electrophoresisin citrate buffer, pH 5.0. No yield or spectroscopic data were given.Thus, the prior art procedure for the synthesis of diuridinetetraphosphate is lengthy and produced only small amounts of onlypartially characterized diuridine tetraphosphate. The present inventionfocuses on methods to produce this medically useful compound which maybe more efficiently and conveniently carried out, and which may beapplied to the large-scale production of diuridine tetraphosphate andsalts thereof.

SUMMARY OF THE INVENTION

The present invention provides new methods for the synthesis of thetherapeutic dinucleotide, P¹, P⁴-di(uridine 5′)-tetraphosphate (FormulaI), and demonstrates applicability to the production of largequantities. The methods of the present invention substantially reducethe time required to synthesize diuridine tetraphosphate, preferably tothree days or less. The ammonium, sodium, lithium, and potassium saltsof P¹, P⁴-di(uridine 5′)-tetraphosphate prepared by these methods arestable, soluble, nontoxic, and easy to handle during manufacture. Thetetrasodium, tetraammonium, tetralithium and tetrapotassium salts of P¹,P⁴-di(uridine 5′)-tetraphosphate (Formula I) prepared by these methodsare highly pure and stable.

wherein:

X is Na, NH₄, Li, K, or H, provided that all X groups are not H.

The method of synthesizing compounds of Formula I, and pharmaceuticallyacceptable salts thereof, is carried out generally by the followingsteps: 1) dissolving uridine or uridine nucleotide compounds of FormulasIIa-d in a polar, aprotic organic solvent and a hydrophobic amine; 2)phosphorylating with a phosphorylating agent of one of the FormulasIVa-b to yield a compound of Formula I, or activating a phosphate groupof the uridine nucleotide compound with an activating agent of one ofthe Formulas IIIa-c and reacting with a suitable compound of Formula IIb-d to yield a compound of Formula I; and 3) purifying by ion exchangechromatography.

Another aspect of the present invention are methods of treating variousdisease states, including, but not limited to: chronic obstructivepulmonary diseases, sinusitis, otitis media, nasolacrimal ductobstruction, dry eye disease, retinal detachment, pneumonia, and femaleinfertility or irritation caused by vaginal dryness.

Another aspect of the present invention is a pharmaceutical compositioncomprising a compound of Formula I together with a pharmaceuticallyacceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides new methods for the synthesis of thetherapeutic dinucleotide, P¹, P⁴-di(uridine 5′)-tetraphosphate, anddemonstrates applicability to the production of large quantities. Themethods of the present invention substantially reduce the time periodrequired to synthesize P¹, P⁴-di(uridine 5′)-tetraphosphate, preferablyto three days or less. The ammonium, potassium, lithium and sodium saltsof P¹, P⁴-di(uridine 5′)-tetraphosphate (Formula I) prepared by thesemethods are stable, soluble, nontoxic, and easy to handle duringmanufacture.

The present invention further provides compounds of Formula I:

wherein:

X is Na, NH₄, Li, K, or H, provided that all X groups are not H.

The sodium, ammonium, lithium and potassium salts of P¹, P⁴-di(uridine5′) -tetraphosphate have many advantages, for example, they provide goodlong-term stability profiles compared to those of divalent cations (e.g.Ca²⁺, Mg²⁺, Mn²⁺) which catalyze hydrolysis of phosphate esters.

These inorganic sodium, ammonium, lithium, and potassium salts impartexcellent water solubility compared to hydrophobic amine salts such astri- and tetrabutylammonium, and similar salts. High water solubility isan important advantage for flexibility in pharmaceutical formulations ofvarying concentration.

The tetrammonium, tetrasodium, tetralithium and tetrapotassium salts ofP¹, P⁴-di(uridine 5′)-tetraphosphate are additionally advantageous inthat they are readily purified by aqueous ion chromatography in which noorganic solvents are used. They have high degree (>90%) of purity, thusare suitable for pharmaceutical use. These tetraammonium and tetramonovalent alkali metal salts are more resistant to hydrolysis than themono-, di-, or tri- salts, therefore, they provide an improved stabilityand a longer shelf life for storage. In addition, these salts are easilyhandled as fluffy, white solids, compared to an oil or gum as with someamine salts.

The tetrasodium, tetralithium, and tetrapotassium salts of P¹,P⁴-di(uridine 5′) -tetraphosphate are non-irritating to the lung andeyes. Other cations may be irritating to the lungs, eyes, and othermucosal epithelia, or are otherwise not well tolerated by the humanbody. The tetrasodium, tetralithium, and tetrapotassium salts arepreferred.

The compounds of Formula I may be used to facilitate the clearance ofmucous secretions from the lungs of a subject such as a mammal includinghumans in need of treatment for various reasons, including cysticfibrosis, chronic bronchitis, asthma, bronchiectasis, post-operativemucous retention, pneumonia, primary ciliary dyskinesia (M. J. Stutts,III, et al, U.S. Pat. No. 5,635,160; PCT International Publication WO96/40059) and the prevention and treatment of pneumonia in immobilizedpatients (K. M. Jacobus and H. J. Leighton, U.S. Pat. No. 5,763,447).Further therapeutic uses include treatment of sinusitis (PCTInternational Publication WO 98/03177), otitis media (PCT InternationalPublication WO 97/29756), dry eye, retinal detachment, nasolacrimal ductobstruction, the treatment of female infertility and irritation due tovaginal dryness via increased mucus secretions and hydration of theepithelial surface, and enhancing the performance of athletes.

The compounds of Formula I may be administered orally, topically,parenterally, by inhalation or spray, intra-operatively, rectally, orvaginally in dosage unit formulations containing conventional non-toxicpharmaceutically acceptable carriers, adjuvants and vehicles. The termtopically as used herein includes patches, gels, creams, ointments,suppositiories, pessaries, or nose, ear or eye drops. The termparenteral as used herein includes subcutaneous injections, intravenous,intramuscular, intrasternal injection or infusion techniques. Inaddition, there is provided a pharmaceutical formulation comprising acompound of general Formula I and a pharmaceutically acceptable carrier.One or more compounds of general Formula I may be present in associationwith one or more non-toxic pharmaceutically acceptable carriers ordiluents or adjuvants and, if desired, other active ingredients. Onesuch carrier would be sugars, where the compounds may be intimatelyincorporated in the matrix through glassification or simply admixed withthe carrier (e.g., lactose, sucrose, trehalose, mannitol) or otheracceptable excipients for lung or airway delivery.

One or more compounds of general Formula I may be administeredseparately or together, or separately or together with: mucolytics suchas DNAse (Pulmozyme®) or acetylcysteine, antibiotics, including but notlimited to inhaled Tobramycin®; non-steroidal anti-inflammatories,antivirals, vaccines, decongestants and corticosteroids.

The pharmaceutical compositions containing compounds of general FormulaI may be in a form suitable for oral use, for example, as tablets,caplets, lozenges, aqueous or oily suspensions, dispersible powders orgranules, emulsion, hard or soft capsules, or syrups or elixirs.Compositions intended for oral use may be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions may contain one or more agentsselected from the group consisting of sweetening agents, flavoringagents, coloring agents and preserving agents in order to providepharmaceutically elegant and palatable preparations. Tablets contain theactive ingredient in admixture with non-toxic pharmaceuticallyacceptable excipients which are suitable for the manufacture of tablets.These excipients may be, for example, inert diluents, such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate; granulating and disintegrating agents, for example, cornstarch, or alginic acid; binding agents, for example, starch, gelatin oracacia; and lubricating agents, for example magnesium stearate, stearicacid or talc. The tablets may be uncoated or they may be coated by knowntechniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate may be employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredients is mixed with water oran oil medium, for example, peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example: sodiumcarboxymethylcellulose, methylcellulose and sodium alginate. Dispersingor wetting agents may be a naturally-occurring phosphatide orcondensation products of an allylene oxide with fatty acids, orcondensation products of ethylene oxide with long chain aliphaticalcohols, or condensation products of ethylene oxide with partial estersfrom fatty acids and a hexitol, or condensation products of ethyleneoxide with partial esters derived from fatty acids and hexitolanhydrides. Those skilled in the art will recognize the many specificexcipients and wetting agents encompassed by the general descriptionabove. The aqueous suspensions may also contain one or morepreservatives, for example, ethyl, or n-propyl p-hydroxybenzoate, one ormore coloring agents, one or more flavoring agents, and one or moresweetening agents, such as sucrose or saccharin.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredients inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example, sweetening, flavoring, and coloringagents, may also be present.

Compounds of Formula I may be administered parenterally in a sterilemedium. The drug, depending on the vehicle and concentration used, caneither be suspended or dissolved in the vehicle. Advantageously,adjuvants such as local anaesthetics, preservatives and buffering agentscan be dissolved in the vehicle. The sterile injectable preparation maybe a sterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent. Among the acceptable vehicles andsolvents that may be employed are sterile water, saline solution, orRinger's solution. The compounds of general Formula I may also beadministered in the form of suppositories for ear, rectal or vaginaladministration of the drug. These compositions can be prepared by mixingthe drug with a suitable non-irritating excipient which is solid atordinary temperatures but liquid at the body temperature and willtherefore melt to release the drug. Such materials are cocoa butter andpolyethylene glycols.

Solutions of compounds of Formula I may be administered byintra-operative installation at any site in the body.

Single dosage levels of the order of from about 1 to about 400 mg,preferably in the range of 10 to 300 mg, and most preferably in therange of 25 to 250 mg, are useful in the treatment of theabove-indicated respiratory conditions. Single dosage levels of theorder of from about 0.0005 to about 5 mg, preferably in the range of0.001 to 3 mg and most preferably in the range of 0.025 to 1 mg, areuseful in the treatment of the above-indicated ophthalmic conditions.The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. It will beunderstood, however, that the specific dose level for any particularpatient will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,sex, diet, time of administration, route of administration, and rate ofexcretion, drug combination and the severity of the particular diseaseundergoing therapy.

The synthetic methods described below encompass several syntheticstrategies for producing P¹, P⁴-di(uridine 5′)-tetraphosphate.Generally, all the methods use uridine or uridine nucleotide compoundsfrom Formula Ila-d as starting materials, which are dissolved in apolar, aprotic organic solvent (e.g. dimethylformamide,dimethylsulfoxide, dioxane, N-methylpyrrolidone, trimethylphosphate) anda hydrophobic amine (e.g. triethylamine, tributylamine, trioctylamine,2,4,6-collidine, tetrabutylammonium, tri- and tetra-alkyl amines,heterocyclic amines). The product is obtained by phosphorylating with aphosphorylating agent from Formula IV (e.g. phosphorus oxychloride,pyrophosphate, pyrophosphorylchloride) or activating a phosphate groupwith an activating agent from Formula III (e.g. carbonyldiimidazole, analky or aryl carbodiimide, an alkyl or aryl phosphochloridate),respectively, with subsequent purification various means well known tothose of skill in the art, including, but not limited to, ionchromatography (e.g. DEAE Sephadex, DEAE cellulose, Dowex 50, anion andcation exchange resins).

The pyrimidine β-D-ribofuranosyl starting materials uridine, uridine5′-monophosphate (UMP), uridine 5′-diphosphate (UDP), and uridine5′-triphosphate (UTP) are shown as free acids in Formulas IIa-d below,respectively. These materials are all commercially available in largequantity in various salt forms.

and salts thereof;

and salts thereof;

and salts thereof.

The activating agents carbodiimide, activated carbonyl, and activatedphosphorus compounds are shown in the general Formulas IIIa-c below,respectively.

wherein R₁and R₂ are C₁-C₈ alkyl or cycloalkyl, C₁-C₈ optionallysubstituted alkyl or cycloalkyl(e.g. hydroxy and amino groups); aryl oroptionally substituted aryl (e.g. hydroxy and amino groups). Preferredcompounds of Formula IIIa are dicyclohexylcarbodiimide and1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

wherein X is imidazole, tetrazole, and/or halogen. Preferred compoundsof Formula IIIb are carbonyldiimidazole and carbonylditriazole.

wherein R₁ and R₂ are C₁-C₈ alkyl or cycloalkyl, C₁-C₈ optionallysubstituted alkyl, alkoxy or cycloalkyl (e.g. hydroxy and amino groups);aryl, alkoxy or optionally substituted aryl or alkoxy (e.g. hydroxy andamino groups) and/or halogen; and X is halogen. Preferred compounds ofFormula IIIc are diphenylphosphorochloridate, phenylphosphorodichloridate, phenylphosphonic dichloride anddiphenylphosphinic chloride.

The mono- and diphosphorylating agents are shown below in the generalformulas IVa-b.

wherein X is halogen. Preferred compound of Formula IVa is phosphorusoxychloride.

wherein X is oxygen, hydroxy, or halogen, and salts thereof. Preferredcompounds of Formula IVb are pyrophosphoryl chloride and pyrophosphate.

Those having skill in the art will recognize that the present inventionis not limited to the following examples and that the steps in thefollowing examples may be varied.

EXAMPLES Example 1 Method for the Production of DiuridineTetraphosphate, Tetrasodium Salt Using Uridine 5′-Diphosphate

Uridine 5′-diphosphate disodium salt (Yamasa, Choshi, Japan; 600 grams)was dissolved in deionized water (5.4 L). The solution was passedthrough a Dowex 50W×4H⁺ (Dow Chemical) column. The fractions containinguridine 5′-diphosphate were pooled and neutralized with tributylamine(Aldrich, St. Louis; 300 mL). The neutralized fractions wereconcentrated to an oil by using a rotary evaporator at a bathtemperature of 55-60° C. The oil was dissolved in dry dimethylformamide(Aldrich, 3 L) and then dried by concentrating to an oil using a rotaryevaporator (55-60° C. bath temperature). This step was repeated twice.The oil was again dissolved in dimethylformamide (3 L) and1,1-carbonyldiimidazole (Aldrich; 100 g) was added. The solution washeated at 50° C. for 2½ hours. An additional amount of activating agent(33 grams) was added and heating continued for a further 2½ hours. Thesolution was again concentrated to an oil on a rotary evaporator (bathtemperature at 55-60° C.). The resulting oil was dissolved in deionizedwater to a conductivity equal to that of 0.2 M NH₄HCO₃. The solution wasthen loaded into a column of Sephadex DEAE-A25 (Pharmacia, Upsala,Sweden; pre-swollen in 1.0 M NaHCO₃ and washed with 2 column volumes ofdeionized H₂O). The column was eluted with the following solutions inthe following order: 60 L of 0.25 M NH₄HCO₃, 120 L of 0.275M NH₄HCO₃, 40L of 0.30 M NH₄HCO₃ and 40 L of 0.35 M NH₄HCO₃. The fractions havingsufficient amounts of pure diuridine tetraphosphate were pooled asdetermined by HPLC analysis and concentrated on a rotary evaporator(bath temperature at 55-60° C.). The resulting residue was dissolved indeionized water (1.5 L) and concentrated on a rotary evaporator. Thisstep was repeated 15 times or until excess of bicarbonate buffer wasremoved. The resulting oil was dissolved in a sufficient amount ofdeionized water to form a ca. 10% solution, the solution charged to aDowex 50W×4 Na⁺(Dow) column and eluted with deionized water. Thefractions containing U₂P₄ were pooled and concentrated to a ca. 10-15%solution, which was lyophilized to yield U₂P₄ tetrasodium salt as awhite solid (150 g approximately 25% yield based on uridine5′-diphosphate).

Structure Elucidation of P¹, P⁴-di(uridine 5′)-tetraphosphate,Tetrasodium Salt

Due to the lack of adequate spectroscopic data of nonadenylateddinucleotides in the literature, a full structure elucidation of P¹,P⁴-di(uridine 5′)-tetraphosphate, tetrasodium salt was performed byemploying modern analytical techniques. The molecular weight wasdetermined by mass spectrometry to be 878 [m/z 855, (M—Na⁺)⁻],confirming the molecular formula C₁₈H₂₂N₄O₂₃P₄.4Na. The exact massmeasured for C₁₈H₂₂N₄O₂₃P₄.3Na [(M—Na⁺)⁻: calculated 854.9318] was854.9268. The measured mass differed from the theoretical mass by 5.0milimass units (5.9 ppm) for a confidence level of 99.7%. Karl Fishermoisture analysis gave a value of 1.73% H₂O and further confirmation ofthe molecular formula was obtained from elemental analysis: calculatedfor Na=10.70, found 10.81%; C:P ratio calculated 1.74, found 1.80, basedon the molecular formula: C₁₈H₂₂N₄O₂₃P₄.4.2Na.1.1H₂O (FW=902.4 g/mol).The infrared spectrum showed a broad signal at 3422 cm⁻¹ and a signal at1702 cm⁻¹, indicating the presence of hydroxyl (O—H stretch) andcarbonyl (C═O stretch) functional groups. In addition, a phosphate P═Ostretch was observed at 1265 cm⁻¹. The UV spectrum in water displayed aλ_(max) of 262 nm with a molar absorptivity of 17,004. The specificrotation at 25° C. (c=1, H₂O) was determined by polarimetry to be−9.56°.

The NMR spectra are: ¹H NMR (D₂O, TMS) δ4.11 (m,2H), 4.14 (m,1H), 4.25(m,1H), 4.27 (m,1H), 5.84 (d, J=8.1 Hz, 1H), 5.86 (d, J=5.4 Hz, 1H),7.81 (d, J=8.1 Hz); ¹³C NMR (D₂O, TMS) δ65.1 (d, J=5.5 Hz), 69.7, 73.5,83.4 (d, J=9.4 Hz), 88.1, 102.8, 141.5, 152.9, 167.5;³¹P NMR (D₂O, H₃PO₄std) δ−22,32 (m),−10.75 (m). The ¹H coupled ⁻P spectrum showed abroadening of the multiplet at δ−10.75 ppm due to the introduction of ¹Hcoupling. This multiplet was therefore confirmed as P_(α). There was noeffect of ¹H coupling on the multiplet at −22.23 ppm, assigning this bydefault as P_(β). A Nuclear Overhauser Effect (NOE) was observed for H₆to the H_(2′) and H_(3′) sugar protons. Because it is not possible forH₅ to show an NOE to the sugar protons, H₆ is confirmed. Additionally,N₁ substitution is confirmed, because no pyrimidine-sugar NOE ispossible for an N₃ substituted structure.

Additional 2-dimensional NMR experiments were conducted to verifyconnectivity. HMQC shows connectivity for H₅ to C₅ and H₆ to C₆,confirming C₅ and C₆. COSY and NOE connectivity were observed for H₅ toH₆, verifying H₅. HMBC 3-bond connectivity was observed for: H₆ toC_(1′), C₆ to H_(1′), H_(1′) to C₂, H₆ to C₂. These data thus confirmH₁, C₂ and N₁ substitution. COSY connectivity of H_(1′) to H_(2′)confirms H_(2′) and HMQC connectivity of H_(1′) to C_(1′) and H_(2′) toC_(2′) confirms C_(1′) and C_(2′). Additionally, HMBC shows 2-bond Jconnectivity from H₅ to C₄, confirming C₄. A ¹³C DEPT spectrum withmult=1.5 shows the carbon at δ65.1 inverted relative to all othercarbons. This observation confirms that C_(5′) is a methylene. Thecoupling of ³¹P to carbons at δ65.1 and 83.4 confirms C_(5′) and C_(4′),because C_(4′) is the only coupled methyne. In addition, HMQC showsconnectivity for C_(5′) to H_(5′) and C_(4′) to H_(4′), confirmingH_(4′) and H_(5′). An NOE was observed for H_(1′) to H_(4′), H₆ toH_(2′) and H₆ to H_(3′), confirming the β anomer sugar configuration.

In conclusion, P¹, P⁴-di(uridine 5′)-tetraphosphate, tetrasodium saltwas synthesized on a 150 g scale in 25% yield from commerciallyavailable starting materials with a total reaction time of 5 hours. Thecrude product was efficiently purified by ion exchange chromatographyand the structure of the reaction product was unambiguously proven usingmass spectroscopic, NMR and other analytical techniques.

Example 2 Method for the Production of Diuridine TetraphosphateTetrammonium Salt Using Uridine 5′-Monophosphate

Uridine 5′-monrophosphate (Sigma, Milwaukee, 3.0 g, 9.26 mmol) wasdissolved in dry DMF (10 mL) and tributylamine (Aldrich, 2 mL). Thesolution was evaporated in vacuo at 40° C. to an oil. The residue wasdissolved in dry DMF (Aldrich, 8 mL) to form a solution.Carbonyldiimidazole (Aldrich, 1.65 g, 10.18 mmol) was added to thissolution. The reaction was heated at 50° C. for one hour. Uridine5′-triphosphate (Yamasa, 5.60 g, 10.18 mmol) prepared as the anhydroustributylammonium salt in DMF (5 mL) and tributylamine (2 mL), asdescribed in Example 3 below, was added to the reaction solution. Themixture was allowed to stir at 50° C. for three days when the solutionwas evaporated in vacuo to an oil, redissolved in water (5 mL) andpurified by column (300×50 mm) chromatography (Sephadex DEAE-A25,40-120μ, Aldrich, pre-swollen in 1.0 M NaHCO₃ and washed with 2 columnvolumes of deionized H₂O) (H₂O→0.3 M NH₄HCO₃ gradient). The purefractions were concentrated in vacuo at 35° C., and H₂O added andreevaporated 5 times to obtain diuridine tetraphosphate tetrammoniumsalt as a white solid (2.37 g, 30% yield): 92.11% pure by HPLC with thesame retention time as the standard. In addition, the tetrammonium saltwas analyzed by FABMS to give a mass of [C₁₈H₂₅N₄O₂₃P₄ (M—H⁺)⁻:calculated 788.9860] 788.9857, confirming a parent formula ofC₁₈H₂₆N₄O₂₃P₄ for the free acid].

Example 3A Method for the Production of Diuridine Tetraphosphate UsingUridine 5′-Triphosphate (UTP)

A solution of uridine 5′-triphosphate (UTP) trisodium salt (ProBioSint,Varese, Italy; 5.86 g, 0.01 mol) in water (5 mL) was passed through acolumn of BioRad AG-MP 50 (Aldrich) strong cation exchange resin in itstributylamine form (50 mL bed volume) and eluted with distilled water(about 300 mL). To this solution was added tributylamine (Aldrich; 5mL), and the suspension shaken until the pH of the aqueous fraction hadrisen to 8. The layers were separated and the aqueous solutionevaporated to small volume, then lyophilized overnight. The residue wasdissolved in dry dimethylformamide (Aldrich; 20 mL) and the solventevaporated at 0.1 mmHg. The dried tributylamine salt was made up to 100mL with anhydrous acetone to yield a stock solution (0.1 M in UTP).Dicyclohexylcarbodiimide (DCC) (Baker, Phillipsburg; 0.227 g, 1.2 mmol)was added to an aliquot of the foregoing UTP solution (10 mL, 1.0 mmol)and the solution stirred at room temperature for 30 minutes. The mixturewas added to the triethylamine salt of uridine 5′-monophosphate (2.0mmol, prepared by addition of triethylamine (0.5 mL) to a solution ofuridine 5′-monophosphate (UMP) (Sigma; 0.648 g in DMF), and evaporatingto dryness). This suspension was then evaporated to dryness, the residuemade up to 5.0 mL in dry DMF, and set aside at 40° C. for 24 hours. Thereaction mixture was separated by semipreparative ion-exchangechromatography (Hamilton PRP X-100 column), eluting with a gradient of0-1.0 M ammonium bicarbonate, 5 mL/min, 30 minutes. The dinucleotidetetraphosphate eluted between 21 and 23 minutes; the product (76.7%yield based on UTP) was quantitated by comparison of its ultravioletabsorption at λ_(max) 263 nm with that of a standard solution of P¹,P⁴-di(uridine 5′)-tetraphosphate.

Example 3B Method for the Production of Diuridine Tetraphosphate UsingUridine 5′-Triphosphate (UTP) and an Excess of Activating Agent

Conversion of UTP to P¹, P⁴-di(uridine 5′)-tetraphosphate can beenhanced by activation of the tributylamine salt (0.1 mmol) with a largeexcess of DCC (0.1 g, 0.5 mmol); in this case the depositeddicyclohexylurea was removed by filtration, the reaction mixtureextracted with ether (10 mL) and the residue dissolved in dry DMF priorto treatment with tributylamine UMP (0.2 mmol). Upon chromatographicseparation of the reaction mixture and quantitation by ultravioletabsorption as in Example 3A above, the uridine tetraphosphate productconstituted 50.7% of the uridylate species in the mixture, correspondingto a conversion from UTP of 95.9%.

Example 4A Method for the Production of Diuridine Tetraphosphate UsingUridine 5′-Monophosphate Activated with Carbonyldiimidazole

Uridine 5′-monophosphate (UMP) (0.324 g, 1.0 mmol) was dissolved in amixture of dry DMF (5 mL) and tributylamine (237 μL, 1 mmol) thesolution was evaporated to dryness, then twice more with DMF to yieldthe anhydrous tributylamine salt. The residue was dissolved in DMF (5mL) and carbonyldiimidazole (CDI) (0.81 g, 5 mmol) added. The solutionwas set aside for 3 hours, then methanol 324 μL, 8 mmol) added todestroy the excess of CDI. The solution was set aside for one hour.Tributylamine pyrophosphate (Sigma, 0.228 g, 0.5 mmol) was added and thesuspension stirred under nitrogen at room temperature. After 3 hours thereaction was quenched with water and the mixture subjected to HPLC as inExample 3A above. Yield of P¹, P⁴-di(uridine 5′)-tetraphosphate asquantitated by its absorbance at 263 nm was 9.3%.

Example 4B Method for the Production of Diuridine Tetraphosphate UsingUridine 5′-Monophosphate Activated with Diphenyl Phosphochloridate

The anhydrous tributylamine salt of UMP (1.0 mmol), prepared essentiallyas above, was dissolved in a mixture of dry dioxane (5 mL) and DMF (1mL). Diphenyl phosphochloridate (0.3 mL) and tributylamine (0.3 mL) wereadded and the solution set aside at room temperature for 3 hours. Thesolvent was evaporated and the residue shaken with ether (˜10 mL), thenset aside at 4° C. for 30 minutes. The ether was decanted and theresidue was dissolved in a solution of tributylamine pyrophosphate(0.228 g, 0.5 mmol) in DMF (3 mL). The solution was stored undernitrogen at room temperature. After 3 hours the reaction was quenchedwith water and the mixture subjected to HPLC as in Example 3A above.Yield of P¹, P⁴-di(uridine 5′)-tetraphosphate as quantified by itsabsorbance at 263 nm was 9.6%.

Example 5 Method for the Production of Diuridine Tetraphosphate UsingUridine, Phosphorus Oxychloride and Pyrophosphate

Uridine (Aldrich, 0.244 g, 1 mmol) was dissolved in trimethyl phosphate(Aldrich, 5 mL) and tributylamine (466 uL, 2 mmol) added. The solutionwas stirred at 0 degrees during the addition of phosphorus oxychloride(0.153 g (93.2 uL), 1 mmol), and the resulting suspension stirred at 0°C. for 3 hours. Tributylamine pyrophosphate (0.228 g) was added and thesuspension stirred at room temperature for 3 hours. The reaction wasquenched with 1.0 M aqueous triethylamine bicarbonate and the mixtureextracted with methylene chloride to remove trimethyl phosphate. Theaqueous solution was subjected to HPLC as in Example 3A above.Conversion of uridine to P¹, P⁴-di(uridine 5′)-tetraphosphate asquantitated by absorbance of the latter at 263 nm was 6.83%.

Example 6 Method for the Production of Diuridine Tetraphosphate UsingUridine 5′-Monophosphate and Pvrophosphoryl Chloride

Uridine 5′-monophosphate (UMP) (64.8 mg, 0.2 mmol) was dissolved in drypyridine (1 mL) and stirred in ice during the addition of pyrophosphorylchloride (13.9 uL (25 mg), 0.1 mmol). The solution became cloudy almostimmediately, then a copious semicrystalline white precipitate formedwhich became a gummy mass within 1-2 minutes. The mixture was stored atroom temperature overnight, then quenched with water and subjected toHPLC as in Example 3A above. Yield of P¹, P⁴-di(uridine5′)-tetraphosphate as quantitated by its absorbance at 263 nm was 15.8%.A substantial amount of P¹, P³-di(uridine 5′-triphosphate) (25.4%) wasobtained as the major by-product.

Example 7 Aqueous Stability and Solubility of P¹, P⁴-di(uridine5′)-tetraphosphate, Tetrasodium Salt

The solubility of P¹, P⁴-di(uridine 5′)-tetraphosphate, tetrasodium saltin water was determined by adding portions of solid to a known volume ofdeionized water until the solution became turbid. The maximum solubilityin water was thus determined to be ca. 900 mg/mL. Stability studies ofthe solid or aqueous solutions incubated at low (5° C.) and elevatedtemperatures (40° C.) showed that less than 1.5% degradation occurs overa three month period as determined by HPLC analysis. The tetrasodiumsalt of P¹, P⁴-di(uridine 5′)-tetraphosphate was thus determined to havean excellent solubility and stability profile suitable forpharmaceutical applications.

Example 8 Toxicity of P¹, P⁴-di(uridine 5′)-tetraphosphate, TetrasodiumSalt in Animals

The nonclinical toxicologic profile of P¹, P⁴-di(uridine5′)-tetraphosphate, tetrasodium salt has been evaluated in a battery ofgenetic toxicology assays that include the bacterial reverse mutationassay, the in vitro mammalian cytogenetic test, the in vitro mammaliancell gene mutation test, and the micronucleus cytogenetic assay in mice.A study in rabbits examined local ocular tolerance and subchronic oculartoxicity after multiple daily administrations over a six-week period. Inaddition, P¹, P⁴-di(uridine 5′)-tetraphosphate, tetrasodium salt hasalso been tested in two single-dose acute inhalation toxicity studies inrat and dog, and one single-dose acute intravenous toxicity study indogs.

The results of these studies show that P¹, P⁴-di(uridine5′)-tetraphosphate, tetrasodium salt is nongenotoxic in a battery ofgenetic toxicology assays. No adverse findings were seen in the oculartoxicology studies. A low degree of acute toxicity was seen in singledose inhalation (rats, dogs) and intravenous (dogs) toxicity studies.P¹, P⁴-di(uridine 5′)-tetraphosphate, tetrasodium salt was thereforedetermined to have an excellent toxicology profile with a wide safetymargin for dosing in humans.

Example 9 Safety and Efficacy of P¹, P⁴-di(uridine 5′)-tetraphosphate,Tetrasodium Salt in Normal Human Volunteers

P¹, P⁴-di(uridine 5′)-tetraphosphate, tetrasodium salt was evaluated ina Phase I, double-blind, placebo-controlled, escalating dose, safety andtolerability study in 75 normal healthy male volunteers. Fortynon-smokers and 35 smokers were evaluated in 5 dosing cohorts of 16volunteers, comprised of 12 receiving a single aerosolized dose of P¹,P⁴-di(uridine 5′)-tetraphosphate, tetrasodium salt (20-400 mg) and 4receiving placebo (normal saline). No serious adverse events werereported. There were no significant changes in FEVI, FVC, MMEF, clinicallaboratory, 12-lead ECG, or urinalysis results in either the placebo oractive drug groups. In smokers, P¹, P⁴-di(uridine 5′)-tetraphosphate,tetrasodium salt produced a 2-fold to 7-fold dose-dependent increase inthe weight of sputum expectorated within 5 minutes of dosing, andstimulation of sputum expectoration was sustained over the next hour ofsputum collection. The effect of P¹, P⁴-di(uridine 5′)-tetraphosphate,tetrasodium salt to induce the expectoration of sputum in non-smokerswas also observed. In conclusion, P¹, P⁴-di(uridine 5′)-tetraphosphate,tetrasodium salt is safe and well-tolerated in normal male subjects andis effective in stimulating the expectoration of sputum when compared toplacebo.

Example 10 Method for the Production of Diuridine Tetraphosphate,Potassium Salt Using Uridine 5′-Diphosphate

Uridine 5′-diphosphate disodium salt is dissolved in deionized water.The solution is passed through a Dowex 50W×4 H⁺ (Dow Chemical) column.The fractions containing uridine 5′-diphosphate are pooled andneutralized with tributylamine (Aldrich, St. Louis, Mo.). Theneutralized fractions are concentrated to an oil form by using a rotaryevaporator at a bath temperature of 55-60° C. The oil is dissolved indry dimethylformamide (Aldrich) and then dried by concentrating to anoil using a rotary evaporator (55-60° C. bath temperature). This step isrepeated. The oil is dissolved in dimethylformamide and1,1-carbonyldiimidazole (Aldrich) is added. The solution is heated at50° C. An additional amount of activating agent is added and heatingcontinued. The solution is again concentrated to an oil form on a rotaryevaporator (bath temperature at 55-60° C.). The resulting oil isdissolved in deionized water. The solution is then loaded into a columnof Sephadex DEAE-A25 (Pharmacia, Upsala, Sweden; pre-swollen in 1.0 MNaHCO₃ and washed with 2 column volumes of deionized H₂O). The column iseluted with the following solutions in the following order: 60 L of 0.25M NH₄HCO₃, 120 L of 0.275M NH₄HCO₃, 40 L of 0.30 M NH₄HCO₃ and 40 L of0.35 M NH₄HCO₃. The fractions having sufficient amounts of purediuridine tetraphosphate are pooled as determined by HPLC analysis andconcentrated on a rotary evaporator (bath temperature at 55-60° C.). Theresulting residue is dissolved in deionized water (1.5 L) andconcentrated on a rotary evaporator. This step is repeated until excessof the bicarbonate buffer is removed. The resulting oil is dissolved ina sufficient amount of deionized waiter to form a ca. 10% solution, thesolution charged to a Dowex 50W×4 H⁺ (Dow) column, which is prewashedwith potassium bicarbonate, and eluted with deionized water. Thefractions containing U₂P₄ are pooled and concentrated, then lyophilizedto yield U₂P₄ tetrapotassium salt as a solid.

Example 11 Method for the Production of Diuridine Tetraphosphate,Lithium Salt Using Uridine 5′-Diphosphate

Uridine 5′-diphosphate disodiuim salt is dissolved in deionized water.The solution is passed through a Dowex 50W×4 H⁺ (Dow Chemical) column.The fractions containing uridine 5′-diphosphate are pooled andneutralized with tributylamine (Aldrich, St. Louis, Mo.). Theneutralized fractions are concentrated to an oil form by using a rotaryevaporator at a bath temperature of 55-60° C. The oil is dissolved indry dimethylformamide (Aldrich) and then dried by concentrating to anoil form using a rotary evaporator (55-60° C. bath temperature). Thisstep is repeated. The oil is dissolved in dimethylformamide and1,1-carbonyldiimidazole (Aldrich) is added. The solution is heated at50° C. An additional amount of activating agent is added and heatingcontinued. The solution is again concentrated to an oil on a rotaryevaporator (bath temperature at 55-60° C.). The resulting oil isdissolved in deionized water. The solution is then loaded into a columnof Sephadex DEAE-A25 (Pharmacia, Upsala, Sweden; pre-swollen in 1.0 MNaHCO₃ and washed with 2 column volumes of deionized H₂O). The column iseluted with the following solutions in the following order: 60 L of 0.25M NH₄HCO₃, 120 L of 0.275M NH₄HCO₃, 40 L of 0.30 M NH₄HCO₃ and 40 L of0.35 M NH₄HCO₃. The fractions having sufficient amounts of purediuridine tetraphosphate are pooled as determined by HPLC analysis andconcentrated on a rotary evaporator (bath temperature at 55-60° C.). Theresulting residue is dissolved in deionized water (1.5 L) andconcentrated on a rotary evaporator. This step is repeated until excessof the bicarbonate buffer is removed. The resulting oil is dissolved ina sufficient amount of deionized water to form a ca. 10% solution, thesolution charged to a Dowex 50W×4 H⁺ (Dow) column, which is prewashedwith lithium carbonate, and eluted with deionized water. The fractionscontaining U₂P₄ are pooled and concentrated, then lyophilized to yieldU₂P₄ tetralithium salt as a solid.

Example 12 Solubility and Stability of P¹, P⁴-di(uridine5′)-tetraphosphate, Tetralithium Salt

The solubility of P¹, P⁴-di(uridine 5′)-tetraphosphate, tetralithiumsalt in water is determined by adding portions of solid to a knownvolume of deionized water until the solution becomes turbid. Stabilitystudies are performed by incubating the solid or aqueous solutions atlow (5° C.) and elevated temperatures (40° C.) for a period of time. Thetetralithium salt of P¹, P⁴-di(uridine 5′)-tetraphosphate is determinedto have an excellent solubility and stability profile suitable forpharmaceutical applications.

Example 13 Solubility and Stability of P¹, P⁴-di(uridine5′)-tetraphosphate, Tetrapotassium Salt

The solubility of P¹, P⁴-di(uridine 5′)-tetraphosphate, tetrapotassiumsalt in water is determined by adding portions of solid to a knownvolume of deionized water until the solution became turbid. Stabilitystudies are performed by incubating the solid or aqueous solutions atlow (5° C.) and elevated temperatures (40° C.) for a period of time. Thetetrapotassium salt of P¹, P⁴-di(uridine 5′)-tetraphosphate isdetermined to have an excellent solubility and stability profilesuitable for pharmaceutical applications.

Example 14 Toxicity of P¹, P⁴-di(uridine 5′)-tetraphosphate,Tetralithium Salt in Animals

The nonclinical toxicologic profile of P¹, P⁴-di(uridine5′)-tetraphosphate, tetralithium salt is evaluated in a battery ofgenetic toxicology assays that include the bacterial reverse mutationassay, the in vitro mammalian cytogenetic test, the in vitro mammaliancell gene mutation test, and the micronucleus cytogenetic assay in mice.A study in rabbits examines local ocular tolerance and subchronic oculartoxicity after multiple daily administrations over a six-week period. Noadverse findings are seen in the ocular toxicology studies. The P¹,P⁴-di(uridine 5′)-tetraphosphate, tetralithium salt has an excellenttoxicology profile with a wide safety margin.

Example 15 Toxicity of P¹, P⁴-di(uridine 5′)-tetraphosphate,Tetrapotassium Salt in Animals

The nonclinical toxicologic profile of P¹, P⁴-di(uridine5′)-tetraphosphate, tetrapotassium salt is evaluated in a battery ofgenetic toxicology assays that include the bacterial reverse mutationassay, the in vitro mammalian cytogenetic test, the in vitro mammaliancell gene mutation test, and the micronucleus cytogenetic assay in mice.A study in rabbits examines local ocular tolerance and subchronic oculartoxicity after multiple daily administrations over a six-week period. Noadverse findings are seen in the ocular toxicology studies. The P¹,P⁴-di(uridine 5′)-tetraphosphate, tetrapotassium salt has an excellenttoxicology profile with a wide safety margin.

What is claimed is:
 1. A P¹, P⁴-di(uridine 5′)-tetraphosphate,tetralithium salt composition having greater than 90% purity.
 2. A P¹,P⁴-di(uridine 5′)-tetraphosphate, tetrapotassium salt composition havinggreater than 90% purity.
 3. The composition according to claim 1,wherein said composition is a solid form.
 4. The composition accordingto claim 1, which is diluted in water.
 5. The composition according toclaim 2, wherein said composition is a solid form.
 6. The compositionaccording to claim 2, which is diluted in water.